This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Fluorescence correlation spectroscopy (FCS) based on statistical analysis of photons emitted by fluorescent particles as they move through a femtoliter observation volume provides quantitative information about mobility of molecules, concentration, composition of molecular complexes, dissociation constants, and reaction kinetics. The established theory of FCS predicts the statistics of photon arrival times based on free diffusion of particles, and particles involved in certain kinds of reactions. We are developing a new method for parameter optimization using virtual FCS in conjunction with Virtual Cell. Based on the molecular mass of each species the diffusion coefficient in the cytoplasm can be predicted. These values can then be used perform a virtual FCS simulation. By integrating the results from multiple species it is possible to calculate a virtual autocorrelation function for the model. Comparison of the virtual autocorrelation function to the experimental FCS autocorrelation function in live cells can be used to optimize parameters in the model. This represents a powerful global approach to parameter optimization in the Virtual Cell.